The present invention is generally in the area of isolating polyesters from plants.
Polyhydroxyalkanoates (PHAs) are a class of naturally occurring polyesters that are synthesized by numerous organisms in response to environmental stress. For reviews, see Byrom, D., "Miscellaneous Biomaterials", in D. Byrom, Ed., "Biomaterials" MacMillan Publishers, London, 1991, pp. 333-359; Hocking, P. J. and Marchessault, R. H., "Biopolyesters," in G. J. L. Griffin, Ed., "Chemistry and Technology of Biodegradable Polymers", Chapman and Hall, London, 1994, pp. 48-96; Holmes, P. A., "Biologically Produced (R)-3-hydroxyalkanoate Polymers and Copolymers," in D. C. Bassett, Ed., "Developments in Crystalline Polymers," Elsevier, London, Vol. 2, 1988, pp. 1-65; Lafferty et al., "Microbial Production of Poly-.beta.-hydroxybutyric acid," H. J. Rehm and G. Reed Eds., "Biotechnology", Verlagsgesellschaft, Weinheim, Vol. 66, 1988, pp. 135-176; Muller and Seebach, Angew. Chem. Int. Ed. Engl., 32:477-502 (1993); and Steinbuchel, A., "Polyhydroxyalkanoic Acids," Byrom, D., Ed., "Biomaterials", MacMillan Publishers, London, 1991, pp. 123-213.
The PHA biopolymers can be divided into two groups according to the length of their side chains (FIG. 1). Those with short side chains (FIG. 1a), such as polyhydroxybutyrate (PHB), a homopolymer of R-3-hydroxybutyric acid units, are crystalline thermoplastics, whereas PHAs with long side chains (FIG. 1b) are more elastomeric. The former have been known for about seventy years (Lemoigne and Roukhelman, Annales des Fermentations, 5:527-536 (1925)) whereas the latter materials were first identified in the early 1980's. De Smet et al., J. Bacteriol., 154:870-878 (1983).
Due to their earlier discovery and their desirable physical properties, the short side chain materials have been more extensively studied. The PHA polymers, which are natural thermoplastics, can be processed using conventional polymer technology and have industrially useful properties, such as biodegradability in soil and marine environments, biocompatibility, and good barrier properties. These characteristics make these materials useful for a wide range of industrial applications.
The PHA polymers may constitute up to 90% of the dry cell weight of bacteria, and are found as discrete granules inside the bacterial cells. These PHA granules accumulate in response to nutrient limitation and serve as carbon and energy reserve materials. Distinct pathways are used by microorganisms to produce each class of these polymers. The pathway leading to the short side chain polyhydroxyalkanoates (SSCPHAs) involves three enzymes, thiolase, reductase and PHB synthase (sometimes called polymerase). Using this pathway, the homopolymer PHB is synthesized by condensation of two molecules of acetyl-Coenzyme A to give acetoacetyl-Coenzyme A, followed by reduction of this intermediate to R-3-hydroxybutyryl-Coenzyme A, and subsequent polymerization (FIG. 2a). The last enzyme in this pathway, the synthase, has a substrate specificity that can accommodate C3-C5 monomeric units including R-4-hydroxy acid and R-5-hydroxy acid units. This biosynthetic pathway is found, for example, in the bacteria Zoogloea ramigera and Alcaligenes eutrophus.
The biosynthetic pathway which is used to make the long side chain polyhydroxyalkanoates (LSCPHAs) is still partly unknown, however, it is currently thought that the monomeric hydroxyacyl units leading to the LSCPHAs are derived by the .beta.-oxidation of fatty acids and the fatty acid pathway (FIG. 2b). The R-3-hydroxyacylcoenzyme substrates resulting from these routes then are polymerized by PHA synthases that have substrate specificities favoring the larger monomeric units in the C6-C14 range. Long side chain PHAs are produced, for example, by Pseudomonads.
The biosynthesis of PHAs has been studied in a wide range of bacteria at both the biochemical and genetic level, and has been reviewed in Steinbuchel et al., FEMS Microbiology Reviews, 103:217-230 (1992). Since the first PHA synthase genes were identified and characterized in 1989 (Peoples and Sinskey, J. Biol Chem., 264:15298-15303 (1989); and U.S. Pat. Nos. 5,229,279, 5,245,023, and 5,250,430 to Peoples and Sinskey), a number of other microbial PHA polymerases have been investigated and their DNA and amino acid sequences published. Steinbuchel et al., FEMS Microbiology Reviews, 103:217-230 (1992). More recently, two subunit PHA synthases from Chromatium vinosum (Liebersgesell, M. and Steinbuchel, A., European J. Biochem., 209:135-150 (1992); and WO 93/02194) and Thiocystis violacea (Liebersgesell, M. and Steinbuchel, A., Appl. Microbiol. Biotechnot. 38:493-501 (1993)) have been described.
The genes encoding the enzymes responsible for the production of SSCPHAs in, for example, Z. ramigera and A. eutrophus, have been isolated and sequenced. Peoples and Sinskey, Prog. Biotechnol. 3:51-56 (1987); Peoples et al., J. Biol. Chem., 262:97-102 (1987); Peoples and Sinskey (1989), J. Biol. Chem. 264:15298-15303, J. Biol. Chem. 264:15293-15297, and Molecular Microbiol. 3:349-357; Stater et al., J. Bacteriol., 170:4431-4436 (1988); and Schubert et al., J. Bacteriol., 170:5837-5847 (1988).
PHA producing microorganisms produce PHA to greater than 60% total dry weight and are readily extractable by organic solvent. Lafferty et al., "Microbial Production of Poly-.beta.-Hydroxybutyric Acid", in H. J. Rehm and G. Reed, Eds., "Biotechnology", Verlagsgesellschaft, Weinheim, Vol. 66, 1988, pp. 135-176. In plants, the extraction and recovery of PHA is significantly complicated by the presence of large amounts of plant oil as well as lower percentages of PHA. These complicating factors make the successful extraction, separation and recovery of PHAs from plants more difficult.
There is a need for the development of methods for the large scale processing and purification of polyhydroxyalkanoates from plant biomass. It is therefore an object of the invention to provide methods for processing PHAs from plant biomass on a large scale. It is another object of the invention to provide methods for isolating PHAs from transgenic oil crop plants. It is a further object of the invention to provide methods for processing plant biomass derived from oil seed crop plants such that the recovery of the non-PHA products such as plant oils also is maximized.